构建重组人pET-32a-瘦素高效的表达载体

Construction of a recombinant pET-32a-leptin efficient expression vector

背景:瘦素的功能是作为脂肪细胞体脂量的信号来认识的,作为体内的能量平衡信号反馈到下丘脑,与其中各部位神经元的受体结合后参与调节进食、饮水、能量代谢。目的:构建原核重组表达载体pET-32a-瘦素并检测其在大肠杆菌BL21中的高效表达。方法:取人脂肪组织,RT-PCR法制备瘦素目的基因,采用DNA重组技术将其克隆至pMD18T与pET-32a原核表达载体,双酶切及测序鉴定,转化大肠杆菌BL21株诱导其表达,SDS-PAGE电泳分离鉴定,表达产物变性复性后,间接酶联免疫吸附法检测抗原反应原性。结果与结论:实验成功扩增出520bp的瘦素目的基因并构建了原核重组表达载体pET-32a-瘦素;测序结果与GenBank收录序列相一致;目的蛋白pET-32a-瘦素呈极高效表达,占总蛋白的50%以上;变性复性后抗原反应原性较复性前明显提高(P<0.01)。结果证实,实验成功构建原核重组高效表达的载体pET-32a-瘦素,并提高其抗原反应原性。

BACKGROUND：Leptin is recognized as a feedback signal of body fat by bonding receptor in the hypothalamus, which is related to ingestion, drinking, and energy metabolism. OBJECTIVE：To construct a prokaryotic recombinant expression plasmid pET-32a-leptin and to analyze its expression in BL21 Escherichia coli. METHODS：The adipose tissues were obtained, and leptin gene was obtained by reverse transcription-PCR. The target gene was orientating cloned into pMD18T and pET-32a vector by DNA recombinant technical. Position clones were transformed into BL21 Escherichia coli and expressed by double digestion and DNA sequencing. Then the expression product was detected for the antigen reactionogenicity after degeneration and renaturation. RESULTS AND CONCLUSION：520 bp leptin fragment was amplified and the prokaryotic recombinant expression plasmid pET-32a-leptin was correctly constructed. The Leptin gene fragment in position clones was tested be same as the sequence of the GenBank public by DNA sequencing. The position protein pET-32a-leptin was highly expressed and about 50% on total protein. The antigen reactionogenicity of expression product was enhanced after degeneration and renaturation （P 0.01）. Prokaryotic recombinant expression plasmid pET-32a-leptin was successfully constructed and the target protein was expressed largely.